Glycogen phosphorylase (EC 2.4.1.1) is a regulatory enzyme whose activity is modulated by a complex mechanism which includes activation by AMP and inhibition by glucose-6-phosphate. An understanding of the interaction between AMP, glucose-6-phosphate, or glucose-1-phosphate and the amino acid residues forming the respective binding sites may shed light on the mechanism of control at a molecular level. The goals of the project are to prepare and characterize nondissociable substrate-activator-, or inhibitor-enzyme complexes by using photoactivatable analogs of the natural substrate and effectors. An azido derivative of AMP, 8-azido-AMP has been tested as an affinity-label for the AMP binding site. The activation of both forms of the enzymes by N3AMP in the absence of AMP, the enhancement of the activity of the enzyme by N3AMP at low concentrations of AMP, and the response of N3AMP both to the a form of the enzyme and to increasing substrate concentration suggest that N3AMP binds to the allosteric site on phosphorylase. Optimum conditions will be established for the incorporation of approximately one mole of analog per mole of phosphorylase monomer. The modified enzyme will be extensively characterized by enzymatic and physical methods and compared to native phosphorylase. Finally, the derivatives will be analyzed chemically; peptides containing the bound analogs will be isolated and sequenced by standard procedures. BIBLIOGRAPHIC REFERENCES: Seery, V.L., Morgan, W.T., and Muller-Eberhard, V. Interaction of rabbit hemopexin with rose bengal and photooxidation of the rose bengal-hemopexin complex. J. Biol. Chem. 250, 6439-6444 (1975).